This invention is directed to novel species-specific malarial polypeptides which are secreted into the plasma of a susceptible mammalian host after infection, and to antibodies directed against those proteins. The polypeptides and/or antibodies are utilized in assays used to diagnose malaria, as well as to determine whether Plasmodium vivax is the species responsible for the infection.
Malaria is transmitted by the bite of the Anopheles mosquito. Minutes after infection, sporozoites (the mosquito-hosted stage of the malarial parasite) enter hepatocytes of the susceptible mammal where they multiply by schizogony and develop into merozoites. Rupture of the infected cells releases the merozoites into the blood, where they enter erythrocytes to begin a phase of asexual reproduction. During acute infections, malarial parasite protein antigens are known to be released, accumulate, and circulate in the plasma of infected individuals (Wilson et al., The Lancet, Jul. 26, 1969; Wilson et al., International Journal for Parasitology 3:511-520, 1973; Wilson et al., Parasitology, 71:183-192; Wilson, Nature, 284:451-452, 1980). The release of these antigens of parasitic origin can occur at the time that infected erythrocytes rupture to allow invasive merozoites to invade new red blood cells. The antigens that spill into the host plasma are those that have accumulated in the host cell cytoplasm and internal membranous structures.
Additionally, release of antigen can occur during the intraerythrocytic growth of the parasite as it matures from the ring stage, the stage which invades the erythrocyte, through the trophozoite stage, and into schizogony when the parasite differentiates into merozoites. Release of antigens at this time involves transport of the protein from the parasite across the parasitophorous vacuole and its membrane, across the host cell cytoplasm to the infected erythrocyte membrane, and then secretion as an intact soluble protein into the plasma of the host. One P. falciparum protein, PfHRP-2 (Histidine Rich Protein-2) has been described that follows this route of transport and is secreted into the culture supernatant or found in plasma (Wellems et al., Proc. Natl. Acad. Sci. USA, 83:6065-6069, 1986; Howard et al., J. of Cell. Biol., 103:1269-1277, 1986; Rock et al., Parasitology, 95:209-227, 1987; Panton et al., Mol. and Biochem. Parasitology, 35:149-160, 1989). A search for HRP analogues in P. vivax using PfHRP gene probes and HRP-antisera gave only negative results (Rock et al., Parasitology, 95:209-227, 1987; J. Barnwell, unpublished results).
There is a need in the field for antibodies specific for a P. vivax blood stage protein in a diagnostic assay. The prior art assays based on antibodies specific for blood stage proteins have been specific only for P. falciparum (Khusmith, Southeast Asian J Trop Med Public Health (THAILAND), 19:21-6, 1988) or have involved the use of panspecies-specific antibodies, so no existing assays are specific for P. vivax (Gao et al., Southeast Asian J Trop Med Public Health (THAILAND), 22:393-6, 1991 and James, MA et al., American Society of Tropical Medicine and Hygiene, Seattle, Wash., Nov. 16-19, 1992, Abs. #135, pp. 145-146). P. vivax has latent liver stages, termed hypnozoites, which are reactivated and reinitiate blood stage parasitemias. Hypnozoites are eliminated by treatment with primaquine, but are not affected by chloroquine, which acts only on blood stage parasites. As P. falciparum does not produce hypnozoites, it is important to identify correctly the Plasmodium species responsible for infection in order to provide the appropriate course of chemotherapy for complete cure. The increased prevalence of drug resistant strains in certain species also makes it important to identify the species involved so correct chemotherapy can be given. Thus, there is a need for a method and reagents adapted. for differential diagnosis of P. vivax malaria.
However, a number of criteria should be met by a particular protein antigen considered as a potential diagnostic target. First, it should be soluble and relatively stable and not rapidly degraded and/or rapidly removed from circulation. Second, the antigen should contain epitopes unique to a species to allow specific diagnosis and preferably be well-conserved within all or most isolates of a species. Additionally, it should be relatively abundant to allow detection at low parasitemia. As discussed below, the proteins of this invention fulfill most or all of these requirements.
Secreted species-specific blood stage antigens have now been identified from a major human malaria parasite species, P. vivax. Two particular such proteins are designated P. vivax Erythrocyte Secreted Protein-1 (PvESP-1) and P. vivax Erythrocyte Secreted Protein-2 (PvESP-2). These antigens and fragments thereof have unique P. vivax-specific epitopes which permits their use in differential determination of P. vivax merozoites. Antibodies can be and have been elicited against unique epitopes of such P. vivax proteins and used in assays which not only diagnose malaria, but also selectively identify P. vivax as the species having caused the infection.